1. Field of the Invention
The present invention relates to methods and kits for the extraction of nucleic acids. In particular, the present invention relates to methods for extracting nucleic acids from tissue samples and paraffin-embedded tissue samples.
2. Background Information
Within the field of biological diagnostics, molecular-based techniques involving the amplification of nucleic acids are being used increasingly for the detection of inherited diseases, cancer, and infectious diseases. However, in applying such amplification techniques with tissues or other important clinical samples, impurities in nucleic acid preparations can inhibit or reduce the sensitivity and efficiency of amplification (Wilson, I. G., Applied and Environmental Microbiology 63(10):3741-3751, 1997).
DNA extraction from archival paraffin-embedded pathology tissue samples is particularly useful in retrospective studies in which the determination of a molecular diagnosis can be correlated with patient outcome. As pointed out by Crisan and Mattson (DNA and Cell Biology 12:455-464, 1993), the advantages of retrospective DNA analysis are multiple and can be applied to: (i) the study of numerous disease processes, where viral, bacterial, or parasitic agents are suspected to play an etiologic role becomes possible and epidemiological or prognostic correlation may be derived; (ii) the study of endogenous DNA abnormalities associated with various types of malignancies; (iii) the study of inherited DNA in genetic diseases; (iv) retrospective studies of rare diseases for which use of archival specimens would allow larger patient study groups than would be possible in prospective studies requiring fresh tissues; and (v),the possibility of correlating the presence or absence of a particular disease, morphological diagnosis or type, disease stage, prognosis, and response to treatment, where the clinical outcome is already known.
Methods reported for the amplification of DNA are based either on the use of a DNA polymerase (e.g., polymerase chain reaction, PCR), a ligase (ligase chain reaction, LCR), or both (GAP-LCR). Of these methods, PCR has been the most widely used to date. PCR involves the hybridization of primers to the strands of a target nucleic acid in the presence of a DNA polymerization agent and deoxyribonucleoside triphosphates under appropriate conditions. The result is the formation of primer extension products throughout several cycles of amplification, and exponential multiplication of the number of original target sequences. Further details about PCR can be obtained by consulting U.S. Pat. No. 4,683,195 (Mullis, et al), U.S. Pat. No. 4,683,202 (Mullis), and U.S. Pat. No. 4,965,188 (Mullis et al).
Because of its inherent sensitivity, product carryover and contamination between samples is a problem with PCR and nucleic acid based amplification systems in general. Product carryover during sample preparation is a serious problem. It is a function of the amount of time that a sample is exposed to the external environment, and related to the number of times the sample containment device must be opened, thereby exposing the sample to the external environment. Thus, it is advantageous to have a sample preparation method that is rapid and allows reduced or minimal exposure of the sample to the external environment; particularly, it is an advantage to provide a method of nucleic acid extraction where the number of times the sample containment device has to be opened is minimal.
Point mutations in the ras proto-oncogenes occur with great frequency in many human cancers, and are a potentially important diagnostic target (Bos, J. L., Cancer Res. 49:4682-4689, 1989). For example, as much as 90% of pancreatic cancer involves a mutation in the K-ras gene; the majority occurring in codon 12 (Almoguera et al., Cell 53:549-554, 1998). There are a number of methods for detecting the presence of a ras mutation. One method, restriction endonuclease mediated selective-PCR (REMS-PCR) has been described recently (WO 9632500). REMS-PCR is based upon the use of a thermostable restriction enzyme during PCR thermocycling. REMS-PCR greatly simplifies and decreases the time required for analysis and detection.
As pointed out by Volenandt et al., the amount of extracted DNA can dramatically affect the yield in a PCR reaction (Polymerase Chain Reaction Analysis of DNA from Paraffin-Embedded Tissue. Methods in Molecular Bioloqy Vol. 15: Current Methods and Applications, 1993, edited by: B. A. White, Humana Press Inc., N.J.) When analyzing DNA from fixed tissue, an inverse relationship between the volume of extracted sample added and PCR amplification yield often is observed. This is due to the effects of certain fixatives and other inhibitors on Taq DNA polymerase activity. Furthermore, nucleic acid fragmentation occurring during fixation or DNA extraction also can be a problem in amplification of DNA (Greer et al., Am. J. Clin. Pathol. 95:117-124, 1991; and Crisan et al., Clin. Biochem. 25:99-103, 1992).
In the case of DNA extraction from paraffin or fresh tissue sections, complex methods for DNA preparation are typically used. Such methods require long incubations with protease enzymes in the presence of surfactants to release DNA and to degrade proteins that can interfere in nucleic acid amplification. Other subsequent steps in purification of extracted DNA may include treatment with an RNAase to remove contaminating RNA, followed by DNA precipitation with a solvent such as ethanol or a mixture of solvents such as phenol and isoamyl alcohol to remove protein and other cellular material, followed by DNA hydration (Volenandt et al., Polymerase Chain Reaction Analysis of DNA from Paraffin-Embedded Tissue. Methods in Molecular Biology Vol. 15: Current Methods and Applications, 1993, edited by: B. A. White, Humana Press Inc., N.J.). In the case of paraffin-embedded tissues, paraffin is usually removed by extraction with solvents such as xylene in a multiple step procedure prior to the proteinase step. A recent report by Banerjee et al. provides a protocol for DNA release from paraffin-embedded tissues (BioTechniques 18:768-773, 1995). The method involves the following steps: (1) microwave treatment, (2) removal of the paraffin by a centrifugation step, (3) Proteinase K digestion, and (4) a heat step to destroy Protease K activity.
Slebos and his associates have reported a method for releasing DNA from paraffin-embedded tissue which includes the use of three 10 micron sections, an incubation with a non-ionic detergent, and an 18-24 hr incubation with Proteinase K, followed by centrifugation (Diagnostic Molecular Pathology 1(2):136-141, 1992). The resultant supernatant is used directly in PCR amplification.
To overcome the need for long incubation with Proteinase K and the need for a heat inactivation step, the provisional specification of NZ 233270 describes the use of a thermostable proteinase instead of Proteinase K for digestion of cell protein and release of nucleic acid. This method provides an improvement in speed and ease-of-use. However, it is limited by the amount of amplifiable DNA that is released from paraffin-embedded tissue. Thus, there is still a need in the art for a rapid and highly effective means of extracting nucleic acids from tissue samples in a manner that is compatible with subsequent amplification procedures.
The present invention overcomes the above-noted problems and provides a needed means of extracting nucleic acids from tissue samples in a manner compatible with subsequent amplification methods. Thus, it is an object of the present invention to provide methods and kits for extracting nucleic acids from tissue samples and paraffin-embedded tissue samples.
Various other objects and advantages of the present invention will be apparent from the detailed description of the invention.
In one embodiment, the present invention relates to a method of extracting nucleic acids from tissue samples. The method comprises contacting the tissue sample with a buffer, at least one nonionic surfactant, and a protease enzyme under conditions sufficient to releases the nucleic acids from the sample. The sample is then heated at an alkaline pH for a period of time sufficient to inactivate the protease enzyme. By centrifuging the sample, the nucleic acids are isolated in the supernatant.
All publications mentioned herein are hereby incorporated by reference.
The present invention relates to methods for extracting nucleic acids from tissue samples so that the extracted nucleic acids are suitable for subsequent amplification and detection using known techniques. Using the present invention, nucleic acids (DNA and/or RNA) can be extracted from human tissue samples, both fresh tissue samples and paraffin-embedded tissue samples. Examples of tissues from which nucleic acids can be extracted using the present invention include, but are not limited to, both normal and cancerous lung tissue, colon tissue, pancreatic tissue, breast tissue, prostate tissue, blood and other body fluids or cellular material containing nucleic acids that can be detected.
In the present invention, tissue samples suspected of containing DNA of interest are contacted with a buffer, at least one nonionic surfactant, and a protease enzyme; sequentially or simultaneously. Suitable common biological buffers include one or more organic buffers that maintain the pH at from about 4 to about 10, and preferably at from about 7 to about 9. Useful buffers include, but are not limited to, 3-(N-morpholino)propanesulfonic acid, 3-(N-morpholino)ethanesulfonic acid, tricine, glycine, TRIS, phosphate, and others readily apparent to those skilled in the art. The amount of buffer used is dependent upon the pKa and is that sufficient to maintain the desired pH.
Any of number of nonionic surfactants can be utilized in the present invention. Examples of surfactants useful in the present invention include, but are not limited to, polyoxyethylenesorbitan derivatives, polyoxyethylene ethers, polyglycol ethers, perfluoroalkyl polyoxyethylenes, fluorinated alkyl alkoxylates and fluorinated alkyl ester compounds. Other useful classes of surfactants and examples of each class would be readily apparent to one skilled in the art, especially after consulting the standard reference for surfactants, McCutcheon""s Emulsfers and Detergents, 1986 North American Edition, McCutcheon Division Publishing Co., Glen Rock, N.J. Representative nonionic surfactants are also provided in U.S. Pat. No. 5,231,015 (Cummins et al.), the contents of which are hereby incorporated by reference. Combinations of nonionic surfactants can also be used in the present invention.
Protease enzyme is used to break down tissues and protein helping to release the nucleic acids. The protease may also degrade nucleases making the released DNA more stable. Preferably, the protease enzyme is thermostable. A wide variety of proteases can be used in the present invention including serine proteases (E. C. 3.4.21, e.g. Typsin, and chymotrypsin), Thiol proteases (E. C. 2.4.22, e.g., papain, ficin), carboxy (acid proteases, E. C. 2.4.23, e.g., pepsin), and metalloproteases (E. C. 2.4.24, e.g.,thermolysin, pronase). Optimum conditions of temperature, pH, ionic strength, and surfactant type may differ depending upon the protease used. Preferred proteinases include Protease K (E. C. 3.4.21.64 from Tritirachium album), protease from Streptomyces griseus (P6911, Sigma Chemical Company, St. Louis), and PRETAQ. PRETAQ is a very thermostable alkaline protease isolated from Thermus sp. Strain RT41 as described by Peek, K., et al. (Purification and characterization of a thermostable proteinase, Eur. J. Biochem 207:1035-1044, 1992). The enzyme is extremely thermostable with no loss of activity reported after 24 hr at 70xc2x0 C., and no loss of activity at room temperature over 6 months. The use of PRETAQ and similar thermostable enzymes have advantages in the preparation of DNA from paraffin tissues since paraffin melts at temperatures around 75xc2x0 C. and above. Thus, the use of such highly thermal stable proteases eliminates the need for separate solvent based extraction protocols for paraffin removal prior to protease digestion. Solvent based de-paraffinization procedures are described in references by Volkenandt et al, Wright and Manos, and Greer, as cited previously, and are based on the use of multiple step treatments with octane or xylene followed by treatment with ethanol or other alcohols, or solvents such as acetone.
The sample is contacted with the buffer, nonionic surfactant and protease enzyme under conditions sufficient to release the DNA. The conditions employed will vary depending on the tissue sample and the protease used but are readily determinable by those skilled in the art. For example, when using PRETAQ protease, the sample can be contacted with the buffer, nonionic surfactant and protease at 70xc2x0 C. to 100xc2x0 C. for 5 minutes to 3 hours depending upon the temperature. At 90xc2x0 C. to 100xc2x0 C., 10-15 minutes is preferred. If Protease K is utilized, the sample can be contacted with the buffer, nonionic surfactant and protease for 30 minutes to 24 hours. Preferably, for 30 minutes to 60 minutes.
The sample is then heated at an alkaline pH for a period of time sufficient to inactivate the protease. The pH of the sample can be adjusted by adding sodium hydroxide or potassium hydroxide to the sample until an alkaline pH is achieved. The extracted nucleic acids can then be isolated by centrifugation.
The method disclosed in the present invention has many advantages. The method is rapid, eliminates the need for potentially toxic solvents, and minimizes the number of manipulations in which the sample containment device has to be opened. The inclusion of a treatment step with heated alkali improves PCR amplification, probably by denaturing the double stranded DNA and converting it into smaller pieces of single-stranded DNA which are more readily amplifiable in PCR. Treatment with hot alkali also is expected to denature the protease, eliminating the concern that residual protease activity would inhibit TAQ polymerase or other enzymes required for subsequent amplification. Thirdly, it is also expected that several chemical agents (heparin, bilirubin, hemoglobin etc.) known to inhibit PCR amplification are denatured by hot alkali treatment. Fourthly, hot alkali treatment destroys RNA which may be an interferent in some amplification protocols. Finally, hot alkali treatment also would be expected to result in solubilization of lipids, fatty acids and other crucial cell membrane. This disruption of the integrity of the cell membrane also would be expected to increase DNA released from cells and tissues.
Nucleic acids extracted from tissue samples according to the present invention are suitable for subsequent amplification using known methods in the art such as PCR and LCR. The general principles and conditions for amplification and detection of nucleic acids using PCR are quite well known, the details of which are provided in numerous references, including U.S. Pat. No. 4,683,195 (Mullis et al.), U.S. Pat. No. 4,683,202 (Mullis), and U.S. Pat. No. 4,965,188 (Mullis et al.), all of which are incorporated herein by reference. Preferably, PCR is carried out using a thermostable DNA polymerase. A number of suitable thermostable DNA polymerases have been reported in the art, including those mentioned in detail in U.S. Pat. No. 4,965,188 (Gelfand et al.) and U.S. Pat. No. 4,889,818 (Gelfand et al.), both incorporated herein by reference. Other reagents that can be used in PCR include, for example, antibodies specific for the thermostable DNA polymerase, which inhibit the polymerase prior to amplification. Such antibodies are represented by the monoclonal antibodies described in U.S. Pat. No. 5,338,671 (Scalice et al.), the contents of which are hereby incorporated by reference.
Amplified nucleic acids can be detected in a number of known ways, such as those described in U.S. Pat. No. 4,965,188 (Gelfand et al.). For example, the amplified nucleic acids can be detected using Southern blotting, dot blot techniques, or nonisotopic oligonucleotide capture detection with a labeled probe. Alternatively, amplification can be carried out using primers that are appropriately labeled, and the amplified primer extension products can be detected using procedures and equipment for detection of the label. Thus, in view of the teachings in the art and the specific teachings provided herein, a worker skilledsin the art should have no difficulty in practicing the present invention to extract nucleic acids from fresh tissue or paraffin-embedded tissue samples, which are suitable for subsequent PCR amplification and detection.
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